U.S. patent application number 13/056631 was filed with the patent office on 2011-09-01 for cvyv-resistant plants of the species cucumis melo.
Invention is credited to Nanne Michiel Faber, Wietsche Kassies, Jacob Pieter Mazereeuw, Teresa Maria Montoro Ponsada, Ana Belen Zapata Barrera.
Application Number | 20110214202 13/056631 |
Document ID | / |
Family ID | 40580771 |
Filed Date | 2011-09-01 |
United States Patent
Application |
20110214202 |
Kind Code |
A1 |
Kassies; Wietsche ; et
al. |
September 1, 2011 |
CVYV-Resistant Plants of the Species Cucumis Melo
Abstract
The present invention relates to Cucumis melo plants that are
resistant against Cucumber Vein Yellowing Virus (CVYV), and to
plant parts derived thereof without the occurrence of necrotic
symptoms. The invention further relates to methods of producing
said plants, to CVYV-resistant plants obtainable by said methods,
as well as to plant parts derived from said CVYV-resistant plants.
Specifically, the present invention relates to a plant of the
species Cucumis melo, comprising in its genome a genetic element,
said genetic element comprising a Cucumber Vein Yellowing Virus
(CVYV)-resistance conferring Quantitative Trait Locus (QTL), or a
CVYV-resistance conferring part thereof, genetically linked to at
least one molecular marker, wherein said marker is selected from a
group consisting of E13/M48-259.69-P2, E16/M48-377.36-P2,
E13/M48-261.30-P2 and E26/M62-076.8-P2.
Inventors: |
Kassies; Wietsche;
(Enkhuizen, NL) ; Montoro Ponsada; Teresa Maria;
(E-Almeria, ES) ; Faber; Nanne Michiel; (Hoorn,
NL) ; Mazereeuw; Jacob Pieter; (Enkuizen, NL)
; Zapata Barrera; Ana Belen; (E-Almeria, ES) |
Family ID: |
40580771 |
Appl. No.: |
13/056631 |
Filed: |
September 2, 2008 |
PCT Filed: |
September 2, 2008 |
PCT NO: |
PCT/EP2008/007214 |
371 Date: |
April 22, 2011 |
Current U.S.
Class: |
800/278 ;
536/23.6; 800/301 |
Current CPC
Class: |
A01H 5/08 20130101 |
Class at
Publication: |
800/278 ;
800/301; 536/23.6 |
International
Class: |
A01H 5/00 20060101
A01H005/00; A01H 5/10 20060101 A01H005/10; A01H 5/08 20060101
A01H005/08; C12N 15/82 20060101 C12N015/82; C07H 21/04 20060101
C07H021/04 |
Claims
1. A plant of the species Cucumis melo, comprising in its genome a
genetic element, said genetic element comprising a Cucumber Vein
Yellowing Virus (CVYV)-resistance conferring Quantitative Trait
Locus (QTL), or a CVYV-resistance conferring part thereof,
genetically linked to at least one molecular marker, wherein said
marker is selected from a group consisting of E13/M48-259.69-P2
(amplification primers SEQ ID Nos: 1 and 2, mobility 259-260 bp),
E16/M48-377.36-P2 (amplification primers SEQ ID Nos: 3 and 4,
mobility 377-378 bp), E13/M48-261.30-P2 (amplification primers SEQ
ID Nos: 5 and 6, mobility 261-262 bp) and E26/M62-076.8-P2
(amplification primers SEQ ID NOs: 7 and 8, mobility 76-77 bp), and
wherein said plant is resistant to CVYV without the occurrence of
necrotic symptoms.
2. Plant according to claim 1, wherein said marker is selected to
the group consisting of E16/M48-377.36-P2 (amplification primers
SEQ ID Nos: 3 and 4, mobility 377-378 bp) and E13/M48-261.30-P2
(amplification primers SEQ ID Nos: 5 and 6, mobility 261-262
bp).
3. Plant according to claim or claim 2, wherein said marker is
E13/M48-261.30-P2 (amplification primers SEQ ID Nos: 5 and 6,
mobility 261-262 bp).
4. Plant according to any of the claims 1 to 3, wherein said
CVYV-resistance conferring QTL, or part thereof, is derived from
melon Cuc6491 with accession number NCIMB 41582.
5. Plant according to any of the claims 1 to 4, wherein said plant
is not melon Cuc6491 with accession number NCIMB 41582.
6. Plant part derived from a plant according to any one of claims
1-5.
7. Plant part according to claim 6, wherein the plant part is a
seed or a fruit.
8. Method for obtaining a CVYV resistant plant of the species
Cucumis melo, comprising introducing a genetic element comprising a
Cucumber Vein Yellowing Virus (CVYV)-resistance conferring
Quantitative Trait Locus (QTL), or a CVYV-resistance conferring
part thereof, genetically linked to at least one molecular marker,
wherein said marker is selected from to the group consisting of
E13/M48-259.69-P2 (amplification primers SEQ ID Nos: 1 and 2,
mobility 259-260 bp), E16/M48-377.36-P2 (amplification primers SEQ
ID Nos: 3 and 4, mobility 377-378 bp), E13/M48-261.30-P2
(amplification primers SEQ ID Nos: 5 and 6, mobility 261-262 bp)
and E26/M62-076.8-P2 (amplification primers SEQ ID NOs: 7 and 8,
mobility 76-77 bp), into the genome of said plant.
9. Method according to claim 8, wherein said marker is selected
from to the group consisting of E16/M48-377.36-P2 (amplification
primers SEQ ID Nos: 3 and 4, mobility 377-378 bp), and
E13/M48-261.30-P2 (amplification primers SEQ ID Nos: 5 and 6,
mobility 261-262 bp).
10. Method according to claim 8 or claim 9, wherein said marker is
E13/M48-261.30-P2 (amplification primers SEQ ID Nos: 5 and 6,
mobility 261-262 bp).
11. Method according to any of the claims 8 to 10, wherein said
CVYV-resistance conferring QTL, or part thereof, is derived from
melon Cuc6491 with accession number NCIMB 41582.
12. Method according to any of the claims 8 to 11, wherein said
plant is not melon Cuc6491 with accession number NCIMB 41582.
13. Cucumber Vein Yellowing Virus (CVYV)-resistant plant of the
species Cucumis melo, obtainable by a method according to any of
the claims 8-12.
14. Use of at least one molecular marker selected from the group
consisting of selected from to the group consisting of
E13/M48-259.69-P2 (amplification primers SEQ ID Nos: 1 and 2,
mobility 259-260 bp), E16/M48-377.36-P2 (amplification primers SEQ
ID Nos: 3 and 4, mobility 377-378 bp), E13/M48-261.30-P2
(amplification primers SEQ ID Nos: 5 and 6, mobility 261-262 bp)
and E26/M62-076.8-P2 (amplification primers SEQ ID NOs: 7 and 8,
mobility 76-77 bp) for obtaining a Cucumber Vein Yellowing Virus
(CVYV)-resistant plant of the species Cucumis melo.
15. Use according to claim 14, wherein the marker is selected from
the group consisting of E16/M48-377.36-P2 (amplification primers
SEQ ID Nos: 3 and 4, mobility 377-378 bp), and E13/M48-261.30-P2
(amplification primers SEQ ID Nos: 5 and 6, mobility 261-262
bp).
16. Use according to claim 14 or claim 15, wherein said marker is
E13/M48-261.30-P2 (amplification primers SEQ ID Nos: 5 and 6,
mobility 261-262 bp).
17. Cucumber Vein Yellowing Virus (CVYV)-resistance conferring
Quantitative Trait Locus (QTL), or a CVYV-resistance conferring
part thereof, genetically linked to at least one molecular marker
selected from the group consisting of E13/M48-259.69-P2
(amplification primers SEQ ID Nos: 1 and 2, mobility 259-260 bp),
E16/M48-377.36-P2 (amplification primers SEQ ID Nos: 3 and 4,
mobility 377-378 bp), E13/M48-261.30-P2 (amplification primers SEQ
ID Nos: 5 and 6, mobility 261-262 bp) and E26/M62-076.8-P2
(amplification primers SEQ ID NOs: 7 and 8, mobility 76-77 bp).
Description
[0001] The present invention relates to Cucumis melo plants that
are resistant against Cucumber Vein Yellowing Virus (CVYV), and to
plant parts derived thereof. The invention further relates to
methods of producing said plants, to CVYV-resistant plants
obtainable by said methods, as well as to plant parts derived from
said CVYV-resistant plants.
[0002] CVYV belongs to the genus Ipomovirus of the family
Potyviridae. It is a rather unstable virus with rod-shaped
particles, generally being 740-800 nm long and 15-18 nm wide. The
viral nucleic acid of CVYV has been reported to consist of double
stranded RNA.
[0003] The virus has a narrow host range that is mainly restricted
to plants of the family of Cucurbitaceae. This family includes
economic important crops like cucumbers, squashes (including
pumpkins), melons and watermelons. Although CVYV infections
generally are a problem in cucumber production, also squash,
zucchini, watermelon and melon productions are affected.
[0004] CVYV is generally transmitted by the whitefly Bremisia
tabaci. CVYV infected plants of the species Cucumis melo (melon)
show vein yellowing or vein clearing and stunting with
corresponding yield reduction. CVYV infection may also cause death
of the plants.
[0005] Thus, CVYV can have disastrous effects in crops when they
become contaminated. Prevention of infection, by, for example,
raising seedlings in a whitefly free environment, or treatment
using for example pesticides, generally is costly and/or unfriendly
to the environment. In addition, these methods do not always
provide satisfactory results.
[0006] No resistance to CVYV has been described in plants of the
species Cucumis melo so far, although several attempts to find such
resistance have been undertaken. Resistance to CVYV has been
reported for other Cucumis species like C. africanus, C. dipsaceus,
and C. prophetarum (Nieto et al. BMC Plant Biology (2007).sub.7:
34). The object of the present invention is to provide plants of
the species Cucumis melo that are resistant to CVYV.
[0007] This object is achieved by the invention by providing a
plant of the species Cucumis melo, comprising in its genome a
genetic element, said genetic element comprising a Cucumber Vein
Yellowing Virus (CVYV)-resistance conferring Quantitative Trait
Locus (QTL), or a CVYV-resistance conferring part thereof,
genetically linked to at least one molecular marker, wherein said
marker is selected from to the group consisting of
E13/M48-259.69-P2 (amplification primers SEQ ID Nos: 1 and 2,
mobility 259-260 bp), E16/M48-377.36-P2 (amplification primers SEQ
ID Nos: 3 and 4, mobility 377-378 bp), E13/M48-261.30-P2
(amplification primers SEQ ID Nos: 5 and 6, mobility 261-262 bp)
and E26/M62-076.8-P2 (amplification primers SEQ ID NOs: 7 and 8,
mobility 76-77 bp), and wherein said plant is resistant to
CVYV.
[0008] According to the present invention the terms
"CVYV-resistant" and "CVYV resistance" relate to the ability of a
plant to restrict the growth and development of the virus under
normal pathogen pressure when compared to non-resistant, i.e.
susceptible plants under similar environmental conditions and pest
pressure.
[0009] The discrimination between resistant and susceptible plants
can, for example, be determined by a method based on mechanical
inoculation. To this end, a CVYV inoculum mixture was prepared by
grinding 10 gram of fresh CVYV infected cucumber (C. sativus L.)
leaves in a phosphate buffer (KH.sub.2PO.sub.4 pH=7.0).
Subsequently, active charcoal and carborundum powder was added to
the mixture. Melon seedlings were dusted with carborundum before
inoculation.
[0010] The term "QTL" is used herein in its art-recognized meaning.
The term "CVYV resistance conferring QTL" refers to a region
located on a particular chromosome of melon that is associated with
at least one gene that encodes for CVYV-resistance or at least a
regulatory region, i.e., a region of a chromosome that controls the
expression of one or more genes involved in CVYV-resistance.
[0011] A QTL may for instance comprise one or more genes of which
the products confer the genetic resistance. Alternatively, a QTL
may for instance comprise regulatory genes or sequences of which
the products influence the expression of genes on other loci in the
genome of the plant thereby conferring the CVYV-resistance.
[0012] According to the present invention, the term "molecular
marker" refers to an indicator that is used in methods for
visualizing differences in characteristics of nucleic acid
sequences. Examples of such indicators are restriction fragment
length polymorphism (RFLP) markers, amplified fragment length
polymorphism (AFLP) markers, single nucleotide polymorphisms
(SNPs), insertion mutations, microsatellite markers,
sequence-characterized amplified regions (SCARs), cleaved amplified
polymorphic sequence (CAPS) markers or isozyme markers or
combinations of the markers described herein which defines a
specific genetic and chromosomal location.
[0013] A "molecular marker linked to a QTL" as defined herein may
thus refer to SNPs, insertion mutations as well as more usual AFLP
markers or any other type of marker used in the field.
[0014] A preferred way to detect the present Cucumber Vein
Yellowing Virus (CVYV)-resistance conferring Quantitative Trait
Locus (QTL), or a CVYV-resistance conferring part thereof using the
present molecular marker is Amplified fragment length polymorphism
(AFLP). AFLP is a Polymerase Chain Reaction (PCR) based genetic
fingerprinting technique that was developed in the early 1990's by
Keygene N. V., Wageningen, The Netherlands.
[0015] In the AFLP method, restriction enzymes are used to cut
genomic DNA, followed by ligation of complementary double stranded
adaptors to the ends of the restriction fragments. A subset of the
restriction fragments are then amplified using primer pairs
complementary to the adaptor and restriction site fragments. The
fragments are visualized, and the mobility or fragment size is
determined relative to a standard DNA ladder to estimate its
molecular weight in base pairs (bp). This can, for example, be done
on denaturing polyacrylamide gels either through autoradiographic
or fluorescence methodologies (Vos et al. Nucleic Acids Res. 1995
23(21):4407).
[0016] The above AFLP method generally results in a multitude of
nucleic acid amplification fragments of different sizes. By
comparing the nucleic acid amplification fragments obtained from,
for example, susceptible and resistant plants, discriminating
nucleic acid amplification fragments, indicating the presence of,
and segregating with, a specific Quantitative Trait Locus, can be
identified. Such identified, and discriminating, amplification
fragments are generally designated as markers of a particular
phenotype, such as a resistant phenotype.
[0017] The present inventors have surprisingly found that plants
comprising in their genome markers E13/M48-259.69-P2 (amplification
primers SEQ ID Nos: 1 and 2, mobility 259-260 bp),
E16/M48-377.36-P2 (amplification primers SEQ ID Nos: 3 and 4,
mobility 377-378 bp), E13/M48-261.30-P2 (amplification primers SEQ
ID Nos: 5 and 6, mobility 261-262 bp) and E26/M62-076.8-P2
(amplification primers SEQ ID NOs: 7 and 8, mobility 76-77 bp) all
had a Cucumber Vein Yellowing Virus (CVYV)-resistance phenotype,
while plants lacking these markers, has a Cucumber Vein Yellowing
Virus (CVYV)-susceptible phenotype. Therefore, the present markers
are genetically linked to, and capable of, identifying, the present
CVYV resistance conferring genetic element.
[0018] According to a preferred embodiment of the present
invention, the markers, linked to the present resistance conferring
Quantitative Trait Locus are selected to the group consisting of
E16/M48-377.36-P2 ((amplification primers SEQ ID Nos: 3 and 4,
mobility 377-378 bp), E13/M48-261.30-P2 (amplification primers SEQ
ID Nos: 5 and 6, mobility 261-262 bp) and E26/M62-076.8-P2
(amplification primers SEQ ID NOs: 7 and 8, mobility 76-77 bp).
[0019] More preferably, the marker, linked to the present
resistance conferring Quantitative Trait Locus, is
E13/M48-261.30-P2 (amplification primers SEQ ID Nos: 5 and 6,
mobility 261-262 bp).
[0020] According to a particularly preferred embodiment, the
CVYV-resistance conferring QTL, or part thereof, is derived from
melon landrace Cuc6491 (accession number NCIMB 41582).
[0021] The term melon accession Cuc6491 is used to indicate the
source of the CVYV-resistance conferring QTL identified herein, and
includes the accession as available from any of the public
collections or depository Institutions well know to the skilled
artisan, as well as CVYV-resistant derivatives thereof.
[0022] Subsequently, said resistance has been introduced into
plants of the species Cucumis melo. According to the invention it
has been found that it was possible to provide fully resistant
plants wherein the present resistance trait, identified in Cuc6491
(accession number NCIMB 41582) has been stably fixed in the genome
of the C. melo plant without the occurrence of necrotic symptoms.
Once inoculated with CVYV, C. melo plants which contain the
appropriate Cuc6491 (accession number NCIMB 41582) segment lack the
typical CVYV mosaic symptoms in contrast to susceptible control
plants.
[0023] The present invention thus also relates to a plant of the
species Cucumis melo, said plant comprising a genetic element, for
example an introgression fragment, comprising a
CVYV-resistance-conferring QTL or a CVYV-resistance-conferring part
thereof, which plant is resistant to CVYV. In a particular
embodiment, the CVYV-resistant plant of the invention is not melon
accession Cuc6491 (accession number NCIMB 41582).
[0024] The term melon accession Cuc6491 (accession number NCIMB
41582) is used to indicate the source of the CVYV-resistance
conferring QTL identified herein, and includes the accession as
available from any of the public collections or depository
institutions well know to the skilled artisan, as well as
CVYV-resistant derivatives thereof.
[0025] The present invention further relates to plant parts derived
from a CVYV-resistant plant of the species Cucumis melo as
described above. The plant part may be any part of a melon plant,
including single cells and cell tissues such as plant cells that
are intact in plants, cell clumps and tissue cultures from which
melon plants can be regenerated. Examples of plant parts include,
but are not limited to, single cells and tissues from pollen,
ovules, leaves, embryos, roots, root tips, anthers, flowers,
fruits, stems shoots, and seeds; as well as pollen, ovules, leaves,
embryos, roots, root tips, anthers, flowers, fruits, stems, shoots,
scions, rootstocks, seeds, protoplasts, calli, and the like.
Preferably, the plant part is a seed or a fruit.
[0026] The present invention furthermore relates to a method for
obtaining a CVYV resistant plant of the species Cucumis melo,
comprising introducing a genetic element comprising a Cucumber Vein
Yellowing Virus (CVYV)-resistance conferring Quantitative Trait
Locus (QTL), or a CVYV-resistance conferring part thereof,
genetically linked to at least one molecular marker, wherein said
marker is selected from to the group consisting of
E13/M48-259.69-P2 (amplification primers SEQ ID Nos: 1 and 2,
mobility 259-260 bp), E16/M48-377.36-P2 (amplification primers SEQ
ID Nos: 3 and 4, mobility 377-378 bp), E13/M48-261.30-P2
(amplification primers SEQ ID Nos: 5 and 6, mobility 261-262 bp)
and E26/M62-076.8-P2 (amplification primers SEQ ID NOs: 7 and 8,
mobility 76-77 bp), into the genome of said plant.
[0027] According to the present method, the marker is preferably
selected from the group consisting of E16/M48-377.36-P2
(amplification primers SEQ ID Nos: 3 and 4, mobility 377-378 bp),
and E13/M48-261.30-P2 (amplification primers SEQ ID Nos: 5 and 6,
mobility 261-262 bp), and more preferably, the marker is
E13/M48-261.30-P2 (amplification primers SEQ ID Nos: 5 and 6,
mobility 261-262 bp).
[0028] According to a particularly preferred embodiment, the
CVYV-resistance conferring QTL, or part thereof, is derived from
melon Cuc6491 with accession number NCIMB 41582.
[0029] According to yet another preferred embodiment of the present
method, the plant is not melon Cuc6491 with accession number NCIMB
41582
[0030] According to the present invention, a genetic element, i.e.
a nucleic acid (preferably DNA) comprising the QTL of the present
invention or a CVYV-resistance conferring part thereof, is used for
the production of a CVYV-resistant melon plant. Thus, the invention
provides for the use of a QTL of the present invention, or parts
thereof, for producing a CVYV-resistant melon plant, which use
involves the introduction of a nucleic acid sequence comprising
said QTL in a CVYV-susceptible recipient melon plant. Said nucleic
acid sequence may be derived from a suitable CVYV-resistant donor
melon plant.
[0031] Preferably, the genetic element (nucleic acid fragment)
comprising said CVYV-resistance conferring QTL, or part thereof, is
derived from melon accession Cuc6491 (accession number NCIMB
41582). Other related melon plants that exhibit resistance to CVYV
and comprise one or more genes that encode for CVYV resistance may
also be utilized as CVYV-resistance donor plants as the present
invention describes how this material may be identified.
[0032] The introduction of the genetic element, i.e., an (isolated)
nucleic acid comprising the QTL of the invention, or a
resistance-conferring part thereof, into a melon plant may be
performed by any method known to the skilled person. Preferably the
introduction is performed by crossing. In such an embodiment, a
method of the present invention may comprise the step of crossing a
plant of melon accession Cuc6491, or a CVYV-resistant derivative
thereof, with a CVYV-susceptible melon plant; harvesting seed from
the cross, growing said seed to produce Fl progeny plants; selfing
or backcrossing said Fl progeny plants with the donor or recipient
parent to produce F2 seeds; growing said F2 seeds to produce F2
progeny plants; determining the presence in the genome of at least
one of said F2 progeny plants of a CVYV-resistance-conferring QTL,
wherein the presence of said QTL is confirmed by detecting the
presence in the DNA of said at least one F2 progeny plants of a
marker linked to said QTL.
[0033] Said genetic element may furthermore be introduced by
transformation, by protoplast fusion, by a doubled haploid
technique or by embryo rescue or by any other nucleic acid transfer
system, optionally followed by selection of offspring plants
comprising the QTL and exhibiting CVYV-resistance. For transgenic
methods of transfer, a nucleic acid sequence comprising a QTL for
CVYV-resistance according to the present invention, or a
CVYV-resistance-conferring part thereof, may be isolated from a
donor plant by using methods known in the art and the thus isolated
nucleic acid sequence may be transferred to the recipient plant by
transgenic methods, for instance by means of a vector, in a gamete,
or in any other suitable transfer element, such as a ballistic
particle coated with said nucleic acid sequence.
[0034] Plant transformation generally involves the construction of
an expression vector that will function in plant cells. In the
present invention, such a vector comprises a nucleic acid sequence
that comprises a QTL for CVYV-resistance of the present invention,
or a CVYV-resistance-conferring part thereof, which vector may
comprise a CVYV-resistance-conferring gene that is under control of
or operatively linked to a regulatory element, such as a promoter.
The expression vector may contain one or more such operably linked
gene/regulatory element combinations, provided that at least one of
the genes contained in the combinations encodes for
CVYV-resistance. The vector(s) may be in the form of a plasmid, and
can be used, alone or in combination with other plasmids, in a
method for producing transgenic plants that are resistant to CVYV,
using transformation methods known in the art, such as the
Agrobacterium transformation system.
[0035] According to the present invention, the terms "introducing"
or "introgressing" thus refer to both a natural and artificial
process whereby genes, or nucleic acids of one species, variety or
cultivar are transferred into the genome of another species,
variety or cultivar, such as e.g. by crossing those species. The
process may optionally be completed by backcrossing to the
recurrent parent.
[0036] The present invention further relates to a CVYV-resistant
plant of the species Cucumis melo, obtainable by a method as
described above, as well as to any plant parts derived from said
plant, such as, but not limited to, seeds and/or fruits. Said
CVYV-resistant melon plant, or part thereof, thus comprises within
its genome a QTL associated with CVYV-resistance, or a
CVYV-resistance-conferring part thereof, as defined herein above,
derived from melon accession Cuc6491 (accession number NCIMB
41582), or a CVYV-resistant derivative thereof, wherein said QTL or
said CVYV-resistance conferring part thereof is not in its natural
genetic background. Such a plant may be obtained by using various
methods well known in the art, either transgenic or
non-transgenic.
[0037] The present invention is further illustrated in the
following Examples that are not intended to limit the scope of the
invention in any way.
EXAMPLES
Example 1
QTL Mapping for CVYV Resistance in Melon
Introduction
[0038] A F2 population of 300 individuals was constructed from a
cross between a CVYV susceptible Galia line ML100_PI and a CVYV
resistant line Cuc6491 (accession number NCIMB 41582). All 300
plants were phenotyped at two plant development stages for their
CVYV resistance level. Subsequently, markers were identified linked
to QTLs that control the resistance to CVYV, using a QTL mapping
approach.
Materials and Methods
[0039] Leaf material of 300 F2 individuals of the
ML100.times.Cuc6491 (accession number NCIMB 41582) population as
well as leaf material from both parental lines was obtained.
[0040] The phenotypic data used for QTL analysis consist of the
level of resistance per F2 plant. The phenotype was measured on two
days (May and June 2007) on one site. Two phenotypic classes were
distinguished:
R=resistant S=susceptible
Results
[0041] Forty-eight AFLP primer combinations (PCs) from a EcoRI/MseI
matrix were screened on both parental lines ML 100 and Cuc6491
(accession number NCIMB 41582). The number of markers per PC is
presented in Table 1.
TABLE-US-00001 TABLE 1 The number of markers scored and mapped per
PC in the ML100 .times. Cuc6491 (accession number NCIMB 41582)
genetic map PC KG Markers scored Markers mapped E11/M48 18 18
E11/M58 11 11 E11/M60 15 14 E14/M49 5 5 E14/M51 3 3 E14/M55 6 3
E23/M47 16 15 E23/M48 13 13 E23/M50 14 14 E23/M54 17 17 E23/M55 5 5
E23/M58 14 12 E26/M59 8 5 E26/M60 14 12 E26/M62 9 7 E14/M48 12 11
E11/M49 18 17 E14/M61 12 12 Total 210 194
[0042] The screening resulted in a data set containing 179
co-dominantly scored AFLP markers. A genetic map was constructed
with the use of specialized software (CarteBlanche, Keygene N. V.,
Wageningen, The Netherlands).
[0043] A total of 154 out of the 179 co-dominantly scored markers
were split into 15 linkage groups containing at least 3 markers
with a total map length of 505.7 cM.
[0044] Windows QTL cartographer 2.0. Wang S., C. J. Basten and Z.
B. Zeng; Department of Statistics North Carolina State University,
USA) was used in order to identify QTLs. The analysis was performed
by use of several statistical methods (Single Marker analysis,
Interval Mapping and Composite Interval Mapping).
[0045] Single Marker Analysis (SMA) scans for correlations between
the individual genotypes at marker positions and the corresponding
phenotypical values. For each marker position a likelihood test is
performed. A QTL is found when a peak exceeds the threshold value
that has been declared. For the SMA, a threshold of LOD 3.5 was
chosen.
[0046] Interval Mapping (IM) is an extension of the SMA method. The
IM method uses two observable flanking markers to construct an
interval in which a hypothetical QTL is placed and the likelihood
tests are performed to either confirm or dismiss the QTL. LOD
values are calculated for each incrementing step in the interval.
Also the LOD score profile is calculated for the entire genome. IM
calculates parameters such as the additive and dominant effects,
the explained variance and a number of other parameters for the
position being tested. When a peak exceeds a threshold value
calculated by performing a permutation test the identification of a
QTL is declared.
[0047] Composite Interval Mapping (CIM) performs the same analysis
as the IM method, but takes into account the variance from other
QTLs by including partial regression coefficients from markers in
other regions of the genome. In theory, CIM gives more power and
precision than
Phenotypes Measured in May 2007
[0048] One QTL was detected. Associations between marker scores and
phenotypes were detected when IM and CIM were used. A permutation
test was performed to calculate the LOD significance thresholds for
phenotypic data (IM: threshold LOD=3.91 and CIM: threshold
LOD=3.97). The best marker, E26/M62-076.8 explains 19.2% of the
variance with a LOD value of 4.81 in this subset of the F2
population (CIM).
Phenotypes Measured in June 2007
[0049] One QTL was detected. Associations between marker scores and
phenotypes were detected when IM and CIM were used. A permutation
test was performed to calculate the LOD significance thresholds for
phenotypic data (IM: threshold LOD=3.73 and CIM: threshold
LOD=3.60). This analysis confirms the presence of a single locus
involved in CVYV resistance at Linkage Group 9 by means of marker
M1 which explains 10.9% of the variance with a LOD value of 3.82 in
this subset of the F2 population (CIM).
Conclusion
[0050] 179 AFLP markers were scored on 92 F2 individuals of the
ML100 (CVYV susceptible).times.Cuc6491 (accession number NCIMB
41582) (CVYV resistant) F2 population. A genetic map was
constructed containing 154 AFLP markers consisting of 15 linkage
groups with a total map length of 505.7 cM.
[0051] QTL mapping using phenotypic data resulted in the
identification of one CVYV QTL. This QTL was mapped on linkage
group 9. For the first phenotypic measurement of May 2007 the best
marker is E26/M62-076.8 and explains 19.2% of the variance with a
LOD value of 4.81. For the phenotypic measurement of June 2007, M1
is the best marker which explains 10.9% of the variance with a LOD
value of 3.82.
Example 2
Continuation Mapping of CVYV-Resistance Loci in Melon
Introduction
[0052] A F2 population of 300 individuals was constructed from a
cross between a CVYV susceptible Galia line ML100 and a CVYV
resistant line Cuc6491 (accession number NCIMB 41582). All F2
plants were phenotyped at two plant development stages for CVYV
resistance level. In Example 1, QTL mapping using 92 F2 plants and
F2 phenotype data resulted in the identification of one locus on
Linkage Group 9 of the ML100.times.Cuc6491 (accession number NCIMB
41582) genetic map, which is putatively involved in the control of
CVYV resistance.
[0053] Subsequently, pathotests have been performed on F3 families
of F2 plants. This was aimed at mapping additional loci and
validating the putative locus that was identified in the previous
project by extension of the ML100.times.Cuc6491 (accession number
NCIMB 41582) genetic map and by using the new phenotype data.
Materials
[0054] The biological material of Example 1 was used for this
project; Leaf material of 300 F2 individuals (box nrs: SM07 332,
SMO7 333, SMO7 334 and SMO7 335) of the ML100.times.Cuc6491
(accession number NCIMB 41582) F2 population were used. Also leaf
material was obtained from the parental lines ML100 and Cuc6491.
Additionally, biological material of a germplasm panel that was
phenotyped for CVYV resistance (box NMB07-49) was used in this
example.
[0055] The phenotypic data was generated in 3 different phenotype
trials. The pathotests were executed as follows; [0056] 1) The
first test was executed on F2 plants, which were infected by
natural infection. The phenotype was measured on two dates (May and
June 2007). Two phenotypic classes were distinguished; R=resistant,
and S=susceptible. The phenotypes of both dates were separately
used for identification of CVYV-resistance loci by a QTL mapping
analysis. [0057] 2) The second phenotype trial was executed on F3
families. Depending on the number of available seeds for each F3
family up to 20 F3 individuals per family were infected by natural
infection with CVYV (CVYV_F3 nat). Each F3 plant was scored for the
level of resistance in 2 classes, resistant and susceptible
respectively. [0058] 3) The third test was also executed on F3
families. Depending on the number of available seeds for each F3
family up to 25 F3 individuals per family were infected with CVYV
using a mechanical infection method (CVYV_F3 mec). Also in this
test, each F3 plant was scored for the level of resistance in 2
classes, resistant and susceptible respectively.
Results
Extension of the ML100.times.Cuc6491 (Accession Number NCIMB 41582)
Genetic Map Focusing on the Top of LG 9
[0059] In Example 1, it was found that part of melon Linkage Group
9 was not covered by the ML100.times.Cuc6491 (accession number
NCIMB 41582) genetic map. Furthermore, QTL mapping results suggest
that a CVYV resistance locus is positioned in this region. In order
to validate this, the ML100.times.Cuc6491 (accession number NCIMB
41582) genetic map was extended specifically for this region.
[0060] An additional AFLP screening by using a BSA approach
(Michelmore et al. Proc. Natl. Acad. Sci. USA. (1991) 8(21):9828)
was executed to identify markers specifically for Linkage Group 9.
This screening revealed Markers M2 and M3.
[0061] Subsequently, the two PCs that contain these markers were
screened on the ML100.times.Cuc6491 (accession number NCIMB 41582)
F2 individuals that were used for QTL mapping. Besides the two
markers of special interest 26 additional markers were scored
bringing the total data set on the 92 F2 plants to 210 codominantly
scored markers.
[0062] A new ML100.times.Cuc6491 (accession number NCIMB 41582)
genetic map was constructed according to the method described
above. In this upgrade of the ML100.times.Cuc6491 (accession number
NCIMB 41582) genetic map, a total of 195 out of the 231
co-dominantly scored markers were split into 15 Linkage Groups
containing at least 3 markers with a total map length of 698.2 cM.
The number of Linkage Groups (15) does still not correspond with
the chromosome number of melon (n=12) suggesting that the genome is
still not completely covered by the upgraded version of the
ML100.times.Cuc6491 genetic map.
[0063] Also the size of the current map indicates that a part of
the melon genome is not covered by this map. The size of melon maps
is usually 1000-1500 cM. However, most important for this project
is the extension of Linkage Group 9 of the ML100.times.Cuc6491
(accession number NCIMB 41582) genetic map by 29 cM through the
positioning of markers M2 position on the 0 cM and M3 on 13.4 cM
position.
[0064] After extending the ML100.times.Cuc6491 (accession number
NCIMB 41582) genetic map, new QTL mapping analyses were executed in
order to test the hypothesis of the map positions of the
CVYV-resistance locus in a part of the particular chromosome that
was not covered by the previous version ML100.times.Cuc6491
(accession number NCIMB 41582) genetic map. Also new phenotypic
data on F3 families was included in these QTL analyses.
QTL Mapping
[0065] Windows QTL cartographer software 1 was used in order to
identify CVYV-resistance loci. The analyses were performed by use
of several statistical methods (Single Marker Analysis, Interval
Mapping and Composite Interval Mapping).
[0066] QTL mapping was executed using the phenotype data of F3
families which were infected by natural infection (CVYV_F3 nat) and
using the phenotype data of F3 families after mechanical infection
(CVYV_F3 mec). The average phenotype value per F3 family for each
infection method was used as input file for QTL analyses.
Mapping CVYV-Resistance Loci with F2-Plants Natural CVYV
Infection.
[0067] The results using the F2 phenotypes (Example 1) are briefly
summarized: QTL mapping using the F2 phenotypes of the first date,
May 2007, resulted in the identification of a putative CVYV
resistance locus on the terminal part of Linkage Group 9 of the
ML100.times.Cuc6491 (accession number NCIMB 41582) genetic map. The
best marker is E26/M62-076.8 on 0 cM of the ML100.times.Cuc6491
(accession number NCIMB 41582) genetic map and explains 19.2% of
the variance with a LOD value of 4.81. The same CVYV-resistance
locus was identified using the F2 phenotypes of the second date. No
other CVYV resistance loci were identified using the F2 phenotype
data.
Mapping CVYV-Resistance Locus with F3-Plants Natural CVYV
Infection
[0068] QTL mapping using the F3 phenotypes after natural infection
and using the upgraded version of the ML100.times.Cuc6491 genetic
map resulted in the confirmation of the putative CVYV-resistance
locus on the terminal part of Linkage Group 9 of the
ML100.times.Cuc6491 (accession number NCIMB 41582) genetic map. The
best marker is E26/M62-076.8 on position 29.1 cM of the updated
version of the ML100.times.Cuc 6491 genetic map explains 39.9% of
the variance with a LOD value of 3.8. (note that the cM
position refers to the upgraded version of the ML100.times.Cuc6491
(accession number NCIMB 41582) genetic map which is constructed in
this example). From the QTL LOD plot it can be concluded that the
CVYV resistance on chromosome 9 is covered by the current genetic
map. Mapping CVYV-Resistance Locus with F3-Plants Mechanical CVYV
Infection
[0069] QTL mapping using the F3 phenotypes after mechanical
infection resulted in a further confirmation of the CVYV-resistance
locus on the terminal part of Linkage Group 9 of the
ML100.times.Cuc6491 (accession number NCIMB 41582) genetic map.
[0070] Using the F3 phenotypes after mechanical infection, the best
marker is M3 on 13.4 cM of the updated version of the
ML100.times.Cuc6491 (accession number NCIMB 41582) genetic map and
explains 54.1% of the variance with a LOD value of 9.7 using
Interval Mapping.
[0071] It can be concluded that the CVYV-resistance gene is
positioned between 13 and 37 cM of the upgraded version of the
ML100.times.Cuc6491 genetic map.
Fine Mapping CVYV-Resistance Locus
[0072] A BSA was executed in order to identify additional markers
linked to the CVYV-resistance locus on Linkage Group 9. Forty eight
PCs from the EcoRI/MseI +0/+A matrix were screened on a bulk of 10
plants with a extremely high average F3 phenotype value for both
natural and mechanical infection methods and a bulk of 10 plants
with a extremely low average F3 phenotype value for both natural
and mechanical infection. This BSA resulted in the identification
of 15 candidate linked markers.
[0073] Subsequently, four PCs containing the best candidate markers
were validated on a panel of 48 F2 plants which are recombinant in
the CVYV resistance region of Linkage Group 9. A total of five
markers could be positioned on Linkage Group 9, of which only two
markers map within the CVYV resistance region between 13.4 and 37
cM.
Validation of AFLP Markers Linked to CVYV-Resistance on Linkage
Group 9
[0074] Two PCs containing four AFLP markers linked to the CVYV
resistance locus were screened on a germplasm panel that was
partially phenotyped for CVYV resistance. Marker E26/M62-076.8 has
the best predictive value. This marker scores the Cuc6491
(accession number NCIMB 41582) (=resistant) genotype "+" for 7 out
of 8 resistant plants and it scores the ML100 (=susceptible)
genotype "-" for all susceptible lines and all not phenotyped lines
(which are expected to be susceptible). The predictive value of the
other markers is poor.
Conclusion
[0075] A major QTL for CVYV-resistance from source Cuc6491
(accession number NCIMB 41582) was identified between 13.4 and 37
cM on Linkage Group 9 of the ML100 (=CVYV
susceptible).times.Cuc6491 (accession number NCIMB 41582) genetic
map. Four markers from this region were validated on a germplasm
panel. Marker E26/M62-076.8 has the best predictive value.
Example 3
[0076] The region between 13.4 and 37 cM on Linkage Group 9 of the
ML100 (.dbd.CVYV susceptible).times.Cuc6491 (accession number NCIMB
41582) genetic map was further fine mapped using a marker set
between 15.8 cM and 63.6 cM. The results of this fine mapping are
shown in table 2.
TABLE-US-00002 TABLE 2 Fine mapping of a region between 15.8 and
63.6 cM on Linkage Group 9. E13/ E16/ E13/ E26/ M48- M48- M48- M62-
CVYV 259.69- 377.36- 261.30- 076.8- phenotype P2 P2 P2 P2 R1 R + +
+ + R2 R + + + + R3 R + + + + R4 R + + + + Cuc 6491 R + + + + Cuc
6498 R + + + + Cuc 8632 R + + + - ML 0261 (339.495) S - - - - ML
0269 (339.524) S - - - - ML 0318 (339.517) S - - - - ML 2003
(340.460) S - - - - ML 2004 (330.968) S - - - - ML 2005 (330.969) S
- - - - ML 2006 (330.970) S - - - - ML 2031 (339.480) S - - - - ML
2020 (339.445) S - - - - ML 2027 (339.477) S - - - - ML 2038
(339.395) S - - - - ML 2039 (339.437) S - - - - ML 2042 (339.443) S
- - - - ML 2043 (339478) S - - - - ML 2064-5 S - - - - ML 2064-11 S
- - - - ML 2066B S - - - - ML 2067-1 S - - - - ML 2067-2 S - - - -
ML 2067-7 S - - - - ML 2067-8 S - - - - S0610031-01 S - - - -
S0610032-04 S - - - - S0610034-04 S - - - - S0610039-02 S - - - -
S0610040-14 S - - - - S0610041-11 S - - - - S0610043-02 S - - - -
S0610047-04 S - - - - S0610055-12 S - - - - S0610061-08 S - - - -
S0610172-13 S - - - - S0610173-12 S - - - - S0610177-01 S - - - -
S0610179-05 S - - - - S0513072-3 S - - - - S0513071-13 S - - - -
S0611322-36 S - - - - S05210540-06 S - - - - S0513001-26 S - - - -
S0513021-14 S - - - - S0612398-10 S - - - - S0622061-11 S - - - -
S0622146-12 S - - - - S0622147-04 S - - - - S0622192-13 S - - - -
S0622352-11 S - - - - S0622353-02 S - - - - S0622482-08 S - - - -
S0622116-19 S - - - - S0622128-18 S - - - - PI S - - - - T S - - -
- S S - - - - C S - - - - S0501002 R + + + +
[0077] As can be seen in Table 2, markers E13/M48-259.69-P2
(amplification primers SEQ ID Nos: 1 and 2, mobility 259-260 bp)
and E26/M62-076.8-P2 (amplification primers SEQ ID NOs: 7 and 8,
mobility 76-77 bp) correlated with 7 out of 8 resistant plants
respectively whereas markers E16/M48-377.36-P2 (amplification
primers SEQ ID Nos: 3 and 4, mobility 377-378 bp) and
E13/M48-261.30-P2 (amplification primers SEQ ID Nos: 5 and 6,
mobility 261-262 bp) showed a 100% correlation with the resistant
phenotype.
[0078] Markers E13/M48-259.69-P2 (amplification primers SEQ ID Nos:
1 and 2, mobility 259-260 bp), E16/M48-377.36-P2 (amplification
primers SEQ ID Nos: 3 and 4, mobility 377-378 bp),
E13/M48-261.30-P2 (amplification primers SEQ ID Nos: 5 and 6,
mobility 261-262 bp) and E26/M62-076.8-P2 (amplification primers
SEQ ID NOs: 7 and 8, mobility 76-77 bp) mapped on positions 32.6,
32.7, 33.4 and 58.5 cM respectively. These markers are associated
with the CVYV resistance locus with LOD scores of 9.9, 8.4, 9.9 and
4.5 respectively. The sequences of the above mentioned markers are
presented in Table 3:
TABLE-US-00003 TABLE 3 Primer sequences of markers
E13/M48-259.69-P2, E16/M48-377.36-P2, E13/M48-261.30- and
E26/M62-076.8-P2 Mobility (.apprxeq. length in base Marker Primer 1
Primer 2 pairs) E13/M48- GAC TGC GTA GAT GAG TCC 259.69 259.69-P2
CCA ATT CAG TGA GTA ACA C (SEQ ID No: 1) (SEQ ID No: 2) E16/M48-
GAC TGC GTA GAT GAG TCC 377.36 377.36-2P CCA ATT CCC TGA GTA ACA C
(SEQ ID No: 3) (SEQ ID No: 4) E13/M48- GAC TGC GTA GAT GAG TCC
261.30 261.30-2P CCA ATT CAG TGA GTA ACA C (SEQ ID No: 5) (SEQ ID
No: 6) E26/M62- GAC TGC GTA GAT GAG TCC 76.8 076.8-P CCA ATT CTT
TGA GTA ACT T (SEQ ID No: 7) (SEQ ID No: 8)
Sequence CWU 1
1
8118DNAArtificial Sequenceprimer 1 marker E13/M48-259.69-P2
1gactgcgtac caattcag 18219DNAArtificial Sequenceprimer 2 marker
E13/M48-259.69-P2 2gatgagtcct gagtaacac 19318DNAArtificial
Sequenceprimer 1 marker E16/M48-377.36-P2 3gactgcgtac caattccc
18419DNAArtificial Sequenceprimer 2 marker E16/M48-377.36-P2
4gatgagtcct gagtaacac 19518DNAArtificial Sequenceprimer 1 marker
E13/M48-261.30-P2 5gactgcgtac caattcag 18619DNAArtificial
Sequenceprimer 2 marker E13/M48-261.30-P2 6gatgagtcct gagtaacac
19718DNAArtificial Sequenceprimer 1 marker E26/M62-076.8-P
7gactgcgtac caattctt 18819DNAArtificial Sequenceprimer 2 marker
E26/M62-076.8-P 8gatgagtcct gagtaactt 19
* * * * *